C-type inactivation of K+ channels is a molecular process of great physiological significance. In the central nervous system, it affects the firing patterns of neurons on the time-scale of seconds, and impaired/altered inactivation leads to a variety of neurological disorders. In neurons the onset and duration of the conductive state of voltage-gated K+ channels, which are directly modulated by the interplay of activation and inactivation, underlie the action-potential firing rates. In the heart, the extremely fast C-type inactivation of the hERG channel plays an important role in the repolarization of cardiac cells. Thus, understanding the molecular basis of inactivation has a direct impact on human health. The pH-activated bacterial KcsA channel is a critically important prototypical model system; there is strong evidence that the C-type inactivated state of this channel is caused by a constriction of the selectivity filter. Our overarching hypothesis is that C-type inactivation is a conformational change of the selectivity filter controlled by competing factors: local packing and hydrogen bonding interactions establish the inherent thermodynamic stability of the conductive filter, while further stabilization/destabilization is communicated via the allosteric coupling with the intracellular gate, or through- space interactions of the pore domain with the voltage sensor in the case of voltage-activated channels. The goal of the proposed research is to test our hypothesis and delineate the conformational plasticity of the selectivity filter of K+ channels in molecular terms. This will be achieved by relying on a multidisciplinary strategy that combines computational and experimental approaches. In aim 1, we will study the molecular determinants of the activation/inactivation allosteric coupling in the KcsA channel. We will generate an extensive Markov State Model (MSM) encompassing all the microscopic events of opening the intracellular gate, ion conduction, and entry into inactivation on the basis of aggregate data from a large number of unbiased MD trajectories in order to provide a complete computational paradigm of the activation/inactivation gating process in KcsA. We will determine the X-ray structure of KcsA with engineered Shaker-like mutations known to affect C-type inactivation and characterize these systems with MD. In aim 2, we will investigate the structural polymorphism of the selectivity filter in chimeric bacterial channels built from the cationic NaK channel and the calcium-activated cationic channel NaKTs channel. Finally, in aim 3, we will investigate the molecular determinants of activation/inactivation in the voltage-gated K+ channels Shaker, Kv1.2 and hERG using X-ray crystallography, functional measurements, and MD simulations.